A fluffy nanofiber scaffold-based microenvironment enables a simplified, biology-centric scale-up strategy for...

Scalable suspension culture technologies are essential for the large-scale manufacturing of mesenchymal stem/stromal cells (MSCs). However, conventional microcarrier-based systems often fail to achieve scalable efficiency because cell-microcarrier dynamics vary across scales, necessitating complex, scale-dependent agitation protocols. To overcome this limitation, we designed a scale-up-oriented suspension culture system employing fluffy, fibrillated nanofiber scaffolds composed of chitosan and chitin. These nanofibers were readily suspended under continuous, gentle agitation and trapped cells on their surfaces, while spontaneously forming fluffy cell-scaffold aggregates through scaffold agglomeration. The low-adhesive chitosan nanofibers acted as physical spacers that prevented aggregate coalescence, thereby maintaining a microenvironment favorable for proliferation. By defining cell-scaffold aggregate size as the key scaling parameter-a biology-centric approach-, we successfully achieved scale-up from 30 mL to 5 L under continuous gentle agitation, yielding comparable specific growth rates of (3.66 ± 0.28) × 10^-2 h^-1 (30 mL), (3.27 ± 0.40) × 10^-2 h^-1 (1 L), and 3.50 × 10^-2 h^-1 (5 L), and reaching a total yield of 4.23 × 10⁹ cells in the 5-L bioreactor (a single run). These findings demonstrate that fluffy nanofiber scaffolds enable a scale-up strategy that reproduces the cellular microenvironment in a manner that is less affected by scale-dependent physical forces. This concept provides a new framework for designing scalable culture environments applicable not only to cell therapy manufacturing but also to culture supernatant production and cell-based food materials.